Cell growth activating composition containing compound having labdane structure

ABSTRACT

The present invention provides compounds which have, particularly, an inhibitory activity on production of melanin, a cell activating activity and an anti-bacterial activity, are derived from natural sources, and are safe and not harmful. One or more compounds represented by the following general formula are contained:                    
     wherein R 1  represents —CH 2 OH, —COOR 6  or —COOX, whereupon X is a group capable forming a salt and R 6  represents hydrogen or a C 1  to C 3  lower alkyl group; R 2  to R 5  each represent hydrogen or a methyl group; and . . . A . . . represents ═C(CH 3 )—, —C(CH 3 )═, —C(═CH 2 )—, —CH(CH 3 )— or —C(OH)(CH 3 )—.

This application is a division of application Ser. No. 09/294,875, filedApr. 20, 1999.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a biologically active substanceconsisting of specific compounds. Further, the present invention relatesto a melanin production inhibitor, cell growth activator, anti-bacterialagent, etc. containing the above-mentioned compounds. Further, thepresent invention relates to an agent for external application onto theskin, oral cavity compositions, bath additives, etc.

2. Prior Art

Compounds with a wide variety of biological activities have beendeveloped heretofore. In particular there are a large number of reportson compounds derived from natural materials especially from the safetypoint of view.

As typical compounds having biological activity, those having aninhibitory effect on production of melanin are described hereinafter.

As age advances, stains, freckles or deposition of pigment after sunburntends to be formed, increased or settled into the skin. This isproblematic particularly for the middle-aged and the elderly.

Although there are still unrevealed aspects of the mechanism ofgeneration of such acquired pigment (melanin) deposition, it is believedthat melanin pigment formation is caused by hormone abnormality andexternal stimuli such as ultra violet rays from sunlight, oxygen andchemical substances etc., and abnormally deposited in the skin. Thedevelopment of compounds preventing the formation and deposition of thismelanin is highly desired and many compounds have been developedtherefor. These compounds include ascorbic acid and derivatives thereof,a placenta extract, hydroquinone, kojic acid, arbutin and ellagic acid,and further there are a large number of reports on melaninproduction-inhibiting components extracted from plants, and theseinclude a chamomile extract (Matricaria chamomilla L. (Composite))disclosed in Japanese Patent Application Laid-Open No. 8-92056, a goldenflower root extract (Scutellaria baicalensis G. (Labiatae)) disclosed inJapanese Patent Application Laid-Open No. 8-104616, a cumin seed(Cuminum cyminum L. (Umbelliferae)) disclosed in Japanese PatentApplication Laid-Open NO. 8-119848 and a wolo extract (Borassusflabellifera (Palmac)) disclosed in Japanese Patent ApplicationLaid-Open No. 10-29928.

Further, the present inventors also found that a fraction obtained bypurifying solvent extracts from various plants by silica gelchromatography strongly inhibits the production of melanin in B16melanoma cells, and filed for a patent (Japanese Patent Application No.9-254025, Aug. 15, 1997). As a substance having a labdan structure,manool, an extract of Dacrydium biforme is reported to have aninhibitory effect on production of melanin (Japanese Patent ApplicationLaid-Open No. 6-72855) Further the inhibitory effect of derivativesthereof on melanin production is also reported (Japanese PatentApplication Laid-Open Nos. 7-25754, 7-69858, 7-206625 etc.).

However, the majority of these conventional melanin productioninhibitors are inadequate with respect to stability, effects, adverseside effects, etc., so a new melanin inhibitor has been desired.

Cell growth activators are now described hereinafter. In aged skin, theactivity of skin cells is weakened so that wrinkles and flabby skin areformed. Recently, there are many studies in which skin cells themselvesare activated and the function of the skin itself is thus activated toimprove skin conditions, and attention has been paid to the developmentof cell growth activators for activating weakened cells as well as tothe incorporation of such cell activators into an agent for externalapplication onto the skin. Conventional materials used for impartingcell-activating activity include alpha-hydroxy acids such as glycolicacids, single-component materials such as hormones, vitamins,photosensitive elements, allantoin, etc., and extracted componentsincluding animal and plant extracts such as placenta extract,lactobacillus extract, shikon extracts, aloe extract, carrot extract,etc. Further, the present inventors also found that there is strongcell-activating activity in distillated residues of solvent extracts,etc. from various plants, and filed for a patent (Japanese PatentApplication Laid-Open No. 8-284572, Oct. 8, 1996). Further, Labdanumfuranoid diterpenoids (WO 97/45099) are reported as celldifferentiation-inducing materials having a labdan structure. However,the majority of conventional materials and extracts havingcell-activating activity are unsatisfactory with respect to theireffects, so that they have to be applied in a large amount. Also theirstability in storage is not satisfactory. Moreover, they may createsafety problems because of their stimulating properties or similarnature.

Anti-bacterial agents are now described hereinafter. A large number ofmicroorganisms are present on the skin and many of them are notproblematic to a healthy skin, but under bad skin conditions or badgeneral conditions, these microorganisms invade hair follicles, sweatglands and damaged sites to act as causative agents for infections.

In addition, there are some microorganisms which cause body odors ordandruff or oxidize secreted lipids to exert adverse effects ancausative factors for acne. To kill such microorganisms, many compoundshave been used, but many of them are chemically synthesized products, sothere has been a demand for highly safe anti-bacterial agents derivedfrom natural sources.

The anti-bacterial activity of Cistus absolute as one of the extractsfrom Cistus ladaniferus L., Cistus creticus L., Cistus monoperiensis L.,Cistus salvifolius, etc. has already been reported (Nippon KeshohinGijyutusha Kaishi, 27, 227 (1993)), but its active ingredient is notreferred to therein.

SUMMARY OF THE INVENTION

The object of the present invention is to provide compounds which have awide variety of biological activities, are derived from natural sources,and are safe and user-friendly. In particular, the object of the presentinvention is to provide compounds having an inhibitory activity onproduction of melanin, a cell-activating activity and an anti-bacterialactivity.

As a result of their eager study to solve this problem, the presentinventors found that extracts with hot water, or extracts with ethanol,hexane, etc. of stems, branches, leaves, etc. of Cistus ladaniferus L.,Cistus creticus L., Cistus monoperiensis L., Cistus salvifolius , etc.have a strong inhibitory activity on production of melanin, acell-activating activity and an anti-bacterial activity, that theseactions are based on labdanolic acid, and further that labd-7-en-15-oicacid, labd-8(17)-en-15-oic acid, and labd-8-en-15-oic acid obtained bymolecular distillation of the above extracts, or of labdanolic acid havea strong inhibitory activity on production of melanin, a cell-activatingactivity and an anti-bacterial activity. Further, the present inventorsfound that salts thereof or methyl and ethyl ester derivatives thereofand reduced derivatives thereof have also the same activity, and as aresult of additional examination, the present invention was completed atlast. The biologically active substance of the present invention is oneor more compounds represented by the following general formula (1):

wherein R¹ represents —CH₂OH, —COOR⁶, or —COOX, where X is a groupcapable forming a salt and R⁶ represents hydrogen or a C₁ to C₃ loweralkyl group; R² to R⁵ each represent hydrogen or a methyl group; and . .. A . . . represents ═C(CH₃)—, —C(CH₃)═, —C(═CH₂)—, —CH(CH₃)— or—C(OH)(CH₃)—.

In the formula, X includes a group capable of forming a salt such assodium, potassium, ammonium, etc., and R⁶ includes hydrogen, a methylgroup, an ethyl group and a propyl group.

In the present invention, the biologically active substance refers to asubstance having one or more activities selected from an inhibitoryactivity on melanin production, a cell-activating activity and ananti-bacterial activity.

DETAILED DESCRIPTION OF THE INVENTION

The above compounds are those known in the art and their processes arealso known. For example, labdanolic acid is a component in labdanum gumextracted from Cistus ladaniferus (J. Chem. Soc., 1956, 4259-4262), andlabd-8(17)-en-15-oic acid (eperuic acid) and labd-8-en-15-oic acid areobtained by chemically treating labdanolic acid (J. Chem. Soc., 1956,4262-4271). Further, it is reported that eperuic acid is a component ina resin derived from an Eperua falcata tree of the Leguminosae (J. Chem.Soc., 1955, 658-662), and labd-7-en-15-oic acid (cativic acid) is acomponent in a resin from Prioria copaifera G. tree of the Leguminosae(J. Am. Chem. Soc., Vol. 79, 1201-1205, 1957).

However, it was not known which biological activity these substancespossess, neither was it known that they possess an inhibitory activityon production of melanin, a cell-activating activity and ananti-bacterial activity.

Although the plants used for preparing the compounds defined in thepresent invention are not particularly limited insofar as they are plantcontaining said compounds, it is particularly advantageous to employCistus ladaniferus L., Cistus creticus L., Cistus monoperiensis L., andCistus salvifolius plants (Cistaceae family). These are used alone or incombinations thereof. The site of plant used is not particularlylimited, and use is made of leaves, branches, stems, barks, etc. Thesemay be used just after being harvested or after being dried.

Preferably, the method of extracting the desired compounds from saidplants makes use of one or more solvents selected from the groupconsisting of water, lower alcohols, petroleum ether and hydrocarbons.The lower alcohols are those containing 1 to 4 carbon atoms, preferablymethanol, ethanol, etc.

The petroleum ether used may be not only the one known in the art butalso a commercial product.

The hydrocarbon solvents are aliphatic hydrocarbons, alicyclichydrocarbons and aromatic hydrocarbons which are liquid at ordinarytemperatures and, preferably, aliphatic hydrocarbons and aromatichydrocarbons which are liquid at ordinary temperatures, among whichhydrocarbons such as hexane and toluene are particularly preferable.

Although the operation of extraction differs depending on the plant andsolvent used, usually divided pieces of the plant are immersed in thesolvent, optionally under gentle stirring, at room temperature to atemperature of 50° C.

Further, a soxhlet extractor known in the art may also be used.

The time required for extraction is usually 3 to 48 hours.

Alternatively, a method of steam distillation or boiling in hot waterafter leaves, branches or stems of the plant are disrupted may also beadopted in the present invention. In this case, gum which floats onwater upon steam distillation or hot-water extraction is removed andthen separated from insolubles by means of solvent extraction.

Further, commercially available products obtained from the above plantsby any of the methods described above may be used.

The crude extract thus obtained contains 25 to 35% labdanolic acid. Thiscrude extract itself may be used as a melanin production inhibitor, cellactivator and anti-bacterial agent.

Hereinafter, a typical method of obtaining the acid or a mixture of theacids from the crude extract or from a commercially available extract isdescribed, but the present invention is not limited to this example.

The above crude extract or a commercially available extract is subjectedto molecular distillation under reduced pressure at 0.1 to 0.5 mm Hgwhereby a fraction at 160 to 230° C. and, preferably, 180 to 220° C. iscollected. This fraction contains a mixture of labd-7-en-15-oic acid,labd-8(17)-en-15-oic acid and labd-8-en-15-oic acid.

As the melanin production inhibitor, cell activator and anti-bacterialagent, this acid mixture may be used as such or if necessary as salts ormethyl or ethyl ester derivatives thereof.

Then, the three acids are separated from this acid mixture.

Specifically, this acid mixture is dissolved in ethanol, then reacted toform ethyl ester derivatives in the presence of a catalytic amount ofsulfuric acid, and subjected to silica gel chromatography on silica geltreated with silver nitrate. The column is washed with hexane and thenthe ethyl ester is eluted with 1% ethyl acetate-hexane. First,labd-8-en-15-oic acid ethyl ester is eluted and then labd-7-en-15-oicacid ethyl ester and labd-8(17)-en-15-oic acid ethyl ester are eluted inthis order. The solvent is distilled off whereby purified products ofthe respective ethyl ester derivatives are obtained. Each of the ethylester derivatives thus obtained is hydrolyzed to give a free acid, andthe free acid is further reacted with diazomethane to give its methylester derivative.

The resulting acids, methyl esters, ethyl esters or mixtures thereof areuseful as melanin production inhibitors, cell activators, andanti-bacterial agents.

Further, these can be incorporated into an agent for externalapplication onto the skin, a bath additive, an oral cavity composition,etc. to give a corresponding agent having an inhibitory activity onproduction of melanin, a cell-activating activity and an anti-bacterialactivity. Further, the compound (1) of the present invention can beadded to prepare an anti-aging agent and anti-wrinkle agent, etc.

The amount of said melanin production inhibitor, cell activator andanti-bacterial agent incorporated is 0.01 to 10% by weight, preferably0.05 to 5% by weight, for the agent for external application onto theskin, 0.1 to 10% by weight, preferably 0.2 to 5% by weight, for the bathadditives, 0.1 to 10% by weight, preferably 0.2 to 5% by weight, for anoral cavity composition, and 0.01 to 5% by weight, preferably 0.05 to 2%by weight, for an anti-bacterial agent.

The amount of the compound (1), if incorporated into face lotion, milkylotion, cream, etc., is usually 0.05 to 10% by weight, preferably 0.05to 2% by weight.

The method of incorporation of the compound (1) into a melaninproduction inhibitor, a cell activator, an anti-bacterial agent, etc. isnot particularly limited. For example, the compound(s) of the inventionmay be incorporated after being diluted with a usual organic solventused in perfumes, that is, ethylene glycol, propylene glycol and loweralcohols which are used alone or as a mixture thereof, or after beingdiluted with a mixture of such solvent and a surface active agent, orafter being mixed with conventional perfume materials. Alternatively, itmay be incorporated as such in the absence of other materials.

Further, the melanin production inhibitor, cell activator,anti-bacterial agent, etc. of the present invention can contain not onlythe above essential ingredients but also other ingredients used inagents for external application onto the skin, such as usual cosmetics,quasi drug preparations, pharmaceutical preparations, etc. For example,it is possible to incorporate skin whitening agents, cell activators,humectants, antioxidants, oil components, surface active agents,thickeners, inorganic fillers, coloring agents, pH adjusters,preservatives, perfumes, UV absorbers, various skin nutrients, etc.,depending on the object or on necessity.

Hereinafter, some of these ingredients are exemplified. As the skinwhitening agents, mention can be made of arbutin, kojic acid, ellagicacid, ascorbic acid, etc. and various derivatives thereof, as well asextracts from various animals and plants such as placenta extract, etc.As the cell activators, mention is made of alpha-hydroxy acids such asglycolic acid, etc., hormones, vitamins, various animal and plantextracts. The humectant includes sorbitol, xylitol, glycerin, propyleneglycol, sodium pyrrolidonecarboxylate, lactic acid, hyaluronic acid,collagen etc.; the antioxidants include vitamin E, butylhydroxytoluene,butylhydroxyanisol etc.; the oil components include vegetable fats andoils such as liquid paraffin, paraffin, olive oil, coconut oil, etc. andanimal fats and oils such as tallow, porcine fat, mink oil, squalane,etc., and synthetic oils such as methyl polysiloxane, silicone oil,glyceryl triisopalmitate, etc.

The surface active agents include anionic surface active agents such assodium lauryl sulfate, triethanolamine laurate, etc., cationic surfaceactive agents such as cetyl trimethyl ammonium chloride, bonzalkoniumchloride, etc., nonionic surface active agents such an glycerylmonoolearate, sorbitan monostearate, polyoxyethylene hydrogenated castoroil, and sucrose fatty acid ester; the thickeners includecarboxymethylcellulose, hydroxyethylcellulose, carboxy-vinylpolymers,sodium alginate carrageenan, etc.; the inorganic fillers include talc,sericite, mica, kaolin, zinc white, titanium oxide, magnesium oxide,etc.; the pH adjusters include organic acids, such as citric acid,sodium citrate, etc. and salts thereof; the preservatives include urea,parabens such as methyl paraben, ethyl paraben, etc., sodium benzoate,ethyl alcohol, etc. Further, by adding various UV absorbers, it is alsopossible to improve the effect of preventing sunburns.

EXAMPLES

Hereinafter, the present invention is described in more detail byreference to the Examples, which are not intended to limit the presentinvention.

Example 1

A commercial labdanum absolute (Givaudan Co., Ltd.) was subjected tomolecular distillation. The labdanum absolute (10 g) was subjected tomolecular distillation under reduced pressure (0.1 mm hg) to collect afraction (4.3 g) at 180 to 220° C. This fraction contains a mixture oflabd-8-en-15-oic acid (Compound 1), labd-7-en-15-oic acid (compound 4)and labd-8(17)-en-15-oic acid (Compound 7) (this mixture is referred tohereinafter as the acid mixture).

The acid mixture (1 g) was dissolved in ether (2 ml) and diazomethanewas added dropwise thereto to give methyl ester derivatives (0.96 g)(the methyl esters are referred to hereinafter as the methyl estermixture).

Similarly, this acid mixture (10 g) was dissolved in ethanol (100 ml)and esterified in the presence of a sulfuric acid catalyst to give ethylester derivatives (9.5 g) (the ethyl ester derivatives are referred tohereinafter as the ethyl ester mixture).

Example 2

For separation of these three acids, the ethyl ester mixture wassubjected to silica gel chromatography. The ethyl ester mixture (10 g)was dissolved in hexane (100 ml) and applied to a column packed withsilica gel treated with silver nitrate and then eluted with a solvent.The eluting solvent firstly used was hexane and then a mixed solvent ofhexane containing 1% by volume of ethyl acetate. Labd-8-en-15-oic acidethyl ester was first eluted, and then labd-7-en-15-oic acid (cativicacid) ethyl ester and labd-8(17)-en-15-oic acid (eperuic acid) ethylester were eluted in this order. The eluate containing each of thecomponents was collected and the solvent was distilled off, wherebypurified products of the respective ethyl ester derivatives (0.83 g,0.16 g and 0.63 g in the order of elution) were obtained. Each of theethyl ester derivatives thus obtained was hydrolyzed to give free acids.

Further, diazomethane was added dropwise to the free acid, and thesolvent was distilled off whereby the methyl ester derivatives wereobtained.

Compound (1): R═H (labd-8-en-15-oic acid)

Compound (2): R═CH₃

Compound (3): R═C₂H₅

Compound (4): R═H (labd-7-en-15-oic acid)

Compound (5): R═CH₃

Compound (6): R═C₂H₅

Compound (7): R═H (labd-8(17)-en-15-oic acid)

Compound (8): R═CH₃

Compound (9): R═C₂H₅

Example 3

The ethyl ester mixture (4.3 g) obtained in Example 1 was dissolved inethanol (10 ml), and 5% palladium carbon catalyst (0.2 g) was addedthereto for hydrogenation reactions to give compound 11 (4.1 g).Further, it was hydrolyzed to give compound 10.

Compound (10): R═H (labden-15-oic acid)

Compound (11): R═C₂H₅

Example 4

The ethyl ester mixture (3.2 g) obtained in Example 1 was dissolved intetrahydrofuran (10 ml) and to this solution a solution of lithiumaluminum hydride (0.21 g) in tetrahydrofuran (10 ml) was added dropwiseat room temperature to give an alcohol derivative mixture (2.32 g),i.e., the terminus —COOH group of labd-8-en-15-oic acid (Compound 1),labd-7-en-15-oic acid (Compound 4) and labd-8(17)-en-15-oic acid(Compound 7) was replaced with —CH₂OH group. This alcohol derivativemixture may be used directly as a melanin production inhibitor, a cellactivator and an anti-bacterial agent.

Test Example 1

Melanin Formation Prevention Test

B16 melanoma cells were suspended (10,000 cells/ml) in DMEM (Dulbecco'smodified eagle medium) containing 10% FBS (fetal bovine serum) and 8 mlof the suspension was added to a cell culture bottle with a 25 cm²bottom surface area, and cultured at 37° C. in the presence of 5% carbondioxide for 3 days. After 3 days of culture, the old medium wasexchanged with 8 ml of fresh medium. 40 micro-liters of a samplesolution, in which compounds of the present invention had been dissolvedin ethanol to give a final concentration shown in the table, werefurther added thereto. As the control, ethanol only was added. After themedium change, the cells were further cultured for 3 days under the sameconditions. After culture was finished, the medium was removed, and thecells were recovered by treatment with trypsin (DIFCO) and suspended in4 ml of phosphate buffered saline (PBS) and a predetermined amount({fraction (1/40)}) of this suspension was used to measure the number ofcells in a Coultar counter (Sysmex Co.) to determine the degree of cellproliferation.

The degree of cell proliferation was determined according to thefollowing equations:

Degree of cell proliferation (%)=(number of cells in sample)/(number ofcells in control)×100

The remainder of the cell suspension was centrifuged and then washedwith 5% trichloroacetic acid, then with ethanol/ethyl ether (3:1 byvolume), and with ethyl ether and the cells were dried, and 2N—NaOH wasadded thereto to dissolve (melanin in) the cells under heating at 70° C.and measured for its optical density (OD) at 420 nm. Using a calibrationcurve of synthetic melanin, the amount of melanin/million cells wasdetermined from the OD and the degree of inhibition of melaninproduction was determined according to the following equation:

Degree of inhibition of melanin (%)=(amount of melanin in control−amountof melanin in sample)/(amount of melanin in control)×100

A degree of inhibition of melanin of 60% or more and a degree of cellproliferation of 70% or more are indicative of superior safety andsuperior inhibitory activity on production of melanin, so that highlypractical usage can be envisaged.

TABLE 1 Degree of Inhibition of Melanin Production Degree of Degree ofConcentration melanin cell Sample (ppm) inhibition proliferation Crude6.3 75% 110% extract Acid mixture 6.3 77% 143% Methyl ester 6.3 80% 122%mixture Ethyl ester 6.3 75% 122% mixture Compound 1 3.1 80% 109%Compound 3 6.3 74% 115% Compound 4 6.3 87% 109% Compound 6 6.3 74% 109%Compound 7 6.3 79% 111% Compound 9 6.3 61% 103% Compound 10 6.3 83%  97%Compound 11 6.3 75% 107% Kojic acid 200.00 34%  94% Arbutin 6.3 74% 100%Ellagic acid 3.1 68%  98%

Test Example 2

Combination Test with Existing Tyrosinase Inhibitors

Using the method of Test Example 1, arbutin, kojic acid, and ellagicacid, which were known to have an inhibitory activity on tyrosinase,were mixed with the compound of the present invention and examined forthe degree of inhibition of melanin production in B16 melanoma cells aswell as for the degree of proliferation of the cells, to determine theeffect of their combined use.

TABLE 2 Effect of Combined use with arbutin Degree of Degree ofConcentration melanin cell Sample (ppm) inhibition proliferation Acid0.4  9% 103% mixture (A) Compound 1 0.4 22% 115% Compound 4 0.4 28% 115%Arbutin (E) 0.8 22%  89% (A) + (E) 0.4 + 0.8 52% 128% (B) + (E) 0.4 +0.8 54% 132% (C) + (E) 0.4 + 0.8 56% 127%

TABLE 3 Effect of combined use with kojic acid Degree of Degree ofConcentration melanin cell Sample (ppm) inhibition proliferation Acid0.4 37% 114% mixture (A) Compound 1(B) 0.4 46% 122% Compound 4(C) 0.451% 119% Compound 7(D) 0.4 34% 130% Kojic Acid (E) 200 34%  94% (A) +(E) 0.4 + 200 55%  96% (B) + (E) 0.4 + 200 43%  96% (C) + (E) 0.4 + 20071%  96% (D) + (E) 0.4 + 200 54% 113%

TABLE 4 Effect of combined use with ellagic acid Degree of Degree ofConcentration melanin cell Sample (ppm) inhibition proliferation Acid0.4 17% 106% mixture (A) Compound 7 (B) 0.4 30% 111% Ellagic Acid (C)1.6 17%  98% (A) + (C) 0.4 + 1.6 31% 111% (B) + (C) 0.4 + 1.6 31% 100%

As can be seen from Tables 2, 3 and 4, when arbutin, kojic acid, andellagic acid were used with the compound of the present invention, theyshowed higher inhibitory activity on melanin production than when theywere used alone. The degree of cell proliferation was also higher thanwhen these tyrosinase inhibitors were used alone. This indicates thatthese materials used in combination with the compounds of the presentinvention bring about a higher effect as skin whitening agents.

Test Example 3

Effect of Diminishing UV Ray-induced Pigment Stains in Guinea Pigs

Hair was carefully removed from the back of 5 brown guinea pigs, and ashielding plate provided with four 2.5 cm×2.5 cm openings was attachedto the portion from which hair had been removed, followed by irradiatingit 3 times every second day with UVB rays at an intensity of 450 mj/cm².Immediately after the irradiation of UVB rays, a 70 micro-liters samplewas applied onto the irradiated site once per day for 35 days, and theamount of pigment diminished by this treatment was examined on the daysshown in Table 5. The sample used was an ethanol solution containing 1%acid mixture obtained in Example 4 as the compound of the invention, andethanol only was applied as the control.

For evaluation of the activity, each application site was measured by acalorimeter (CR200b, Minolta Co Ltd.). ΔLx is the difference between theL value after the sample application (value changing with time) and theL value of the portion before the sample application started, and thenΔΔL value was determined by subtracting ΔLo value (i.e. the valuesimilarly determined for the portion to which ethanol was applied) fromthe ΔLx value. The ΔΔL value can be determined according to theequation:

ΔΔL=(Lx−Lo)−(L′x−L′o)

Lo: L value of the test site (site to which the sample was applied)before sample application

Lx : L value of the test site (site to which the sample was applied) onDay x after sample application

L′o : L value of the control site (site to which ethanol was applied)before ethanol application

L′x : L value of the control site (site to which ethanol was applied) onDay x after ethanol application

The same experiment was conducted using an ethanol solution containing7% kojic acid. The results are shown in Table 5.

TABLE 5 Change in ΔΔL value ΔΔL Sample Concentration Day 14 Day 28 Day35 Acid 1% 0.30 2.25 2.15 Mixture Kojic Acid 7% 1.32 1.52 1.40

As can be seen from Table 5, the acid mixture as one of the compounds ofthe present invention has an evident action of diminishing pigmentstains as compared with the control. The activity was stronger than thatof kojic acid after Day 28. When observed with the passage of time,kojic acid was found to indicate the activity at an earlier stage untilDay 14.

Test Example 4

Mushroom Tyrosinase Inhibition Test

Commercial mushroom-derived tyrosinase (Sigma) was used to examineinhibitory activity on tyrosinase. 0.2 ml sample solution was added to2.3 ml phosphate buffer so as to give a final concentration shown inTable 6, then 0.1 ml of a tyrosinase solution (1000 U/ml) was addedthereto, and further 0.4 ml of L-tyrosine solution (0.3 mg/ml) was addedthereto as the substrate, and the mixture was kept at 37° C. for 30minutes. After reaction, the absorbance (OD) at a wavelength of 490 nmwas measured and the degree of inhibition of tyrosinase reaction wasdetermined according to the following equation. For comparison, arbutinand kojic acid known as tyrosinase inhibitors were also examined.

Degree of inhibition (%)=(1−((sample OD−blank OD)/(Sample-free OD−blankOD)))×100

Sample: The buffer, the enzyme solution, the substrate solution and thesample solution

Sample-free: The buffer, the enzyme solution and the substrate solution

Blank: The buffer and the enzyme solution

TABLE 6 Degree of inhibition of mushroom tyrosinase Concentration Degreeof Sample (ppm) inhibition Acid mixture 25 0% Compound 1 25 0% Compound4 25 0% Compound 7 25 0% Arbutin 100  29%  Kojic Acid 25 67% 

As shown in Table 6, arbutin and kojic acid known as tyrosinaseinhibitors inhibited mushroom tyrosinase, while the compounds of thepresent invention as the melanin production inhibitor did not show anyinhibitory activity, thus suggesting the possibility that their activitydoes not lie in the inhibition of tyrosinase.

Test Example 5

Test of Tyrosinase Inhibition in B16 Melanoma Cells

B16 melanoma cells were cultured for 3 days in DMEM containing 10% FBSat 37° C. in the presence of 5% carbon dioxide. The cells proliferatedafter culture were recovered by treatment with trypsin, then suspendedin PBS containing 0.1% Triton X100 at a density of ten million cells/mland disrupted by sonication. The product obtained was centrifuged at11,000 G for 20 minutes, and the resulting supernatant was used as acrude enzyme solution. 0.2 ml sample solution was prepared in aphosphate buffer so as to give a final concentration shown in Table 7.Added thereto was 0.2 ml of crude enzyme solution. The mixture waspreliminarily kept at 37° C. for 5 minutes, and 0.2 ml L-DOPA solution(0.5 mg/ml) was added as the substrate, and the mixture was kept at 37°C. for 3 hours. After 3 hours, the absorbance (OD) at a wavelength of490 nm was measured, and the degree of inhibition of tyrosinase wasdetermined according to the equation shown in Test Example 4. Forcomparisons arbutin and kojic acid known as tyrosinase inhibitors werealso examined.

TABLE 7 Degree of inhibition of tyrosinase in B16 melanoma cellsConcentration Degree of Sample (ppm) inhibition Acid mixture 25 0%Compound 1 25 0% Compound 4 25 0% Compound 7 25 0% Arbutin 100  35% Kojic Acid 25 62% 

As shown in Table 7, arbutin and kojic acid known as tyrosinaseinhibitors inhibited tyrosinase in B16 melanoma cells, while thecompounds of the present invention as the melanin production inhibitordid not exhibit any inhibitory activity.

Test Example 6

Test of Cell Activating Activity

Human-derived normal skin fibroblasts (NB1RGB: Institute of Physical andChemical Research) were suspended into DMEM containing 10% FBS to give aconcentration of 20,000 cells/ml, and 5 ml each of this cellularsuspension was introduced into each of 25 cm² bottles, cultured at 37°C. in the presence of 5% carbon dioxide for 24 hours. To each bottle,was added 10 micro-liter (0.2%) of the ethanol solution containing thecompound to give the final concentration shown in Table 8. The cellswere cultured for an additional 3 days. After 3 days the old medium wasdiscarded and 5 ml fresh medium was added, and the sample was furtheradded thereto. After this exchange of the medium, the cells were furthercultured for another 3 days. Then cells were removed with trypsin andthe number of cells in each bottle was counted with a Coultar counter.

Simultaneously, as the control, only ethanol was added and the NB1RGBcells were cultured and the number of cells were counted in the samemanner.

The number of cells in each bottle to which the sample had been addedwas determined as a relative value to the number (as 100) of cells inthe control after cultured, and the results are shown in Table 8. As thecomparative example, 5 glycolic acid known to have cell activatingactivity was examined and shown in the table.

TABLE 8 Degree cf cell proliferation of fibroblasts Concentration Degreeof cell Sample (ppm) proliferation Crude extract 8.0 130% Acid mixture8.0 130% Methyl ester mixture 4.0 126% Ethyl ester mixture 4.0 110%Compound 3 8.0 134% Compound 6 8.0 134% Compound 7 8.0 128% Compound 98.0 127% Compound 10 8.0 126% Compound 11 8.0 104% Alcohol derivative4.0 138% mixture Glycolic acid 4.0 120%

As shown in Table 8, the compounds of the present invention showed astrong activity of activating proliferation of NB1RGB fibroblasts.

Test Example 7

Anti-bacterial Activity Test

A test was conducted using the 8 aerobic microorganisms and 2 anaerobicmicroorganisms shown in Table 9 below. The aerobic microorganisms wereexamined in an agar medium dilution method and the anaerobicmicroorganisms were examined in a liquid medium dilution method underanaerobic culture conditions.

Agar Medium Dilution Method

Muller Hinton agar medium (DIFCO) was heated and dissolved and 10 ml ofthe medium was introduced into each test tube, sterilized and used. Thesample was prepared by dissolving in ethanol and the resulting ethanolsolution was diluted 2-fold serially with ethanol, and 100 micro-litersof sample solution were added to 10 ml of each dissolved agar medium,then stirred, introduced into a Petri dish of 9 cm in diameter, andsolidified at room temperature. Each test microorganism in a well grownslant was inoculated via one loop of platinum into 10 ml Muller Hintonbroth (DIFCO) and cultured with shaking at 27° C. for 24 hours and usedas a microbial fluid. This fluid was diluted at 108 CFU (Colony formingunit)/ml, and 5 micro-liters of the diluted fluid were inoculated ontothe agar and cultured at 37° C. overnight. Inoculation of themicroorganism was conducted using microplanter MIT-P for 27 tubes testmicroorganisms (Sakuma Seisakusho K. K.).

Judgement of MIC(minimum inhibitory concentration) was conducted bycomparison with the control where the test microorganism was inoculatedand grown onto a Muller Hinton agar plate containing 100 micro-literethanol, and the concentration at which the microorganism did not growwas regarded as MIC.

Liquid Medium Dilution Method

10 ml GAM broth (Nissui) was introduced into each test tube equippedwith a cap and sterilized. 100 micro-liter of the sample solutionprepared for agar medium dilution method was added to each test tube andlightly stirred, and 100 micro-liter test microbial solution (10⁷CFU/ml) was added thereto, then capped and incubated at 37° C. The testmicrobial solution was prepared by introducing 100 micro-liter stockmicrobial solution into 10 ml GAM broth in a test tube then capping thetube and fermenting it at 37° C. overnight.

Judgement of MIC was conducted by comparison with the control to which100 micro-liter ethanol was added, and the concentration at which themicroorganism did not grow was regarded as MIC.

TABLE 9 Test Microorganisms Test Microorganism Code Aerobicmicroorganisms: Staphylococcus epidermidis JCM 2414 Se-1 Staphylococcusepidermidis var. H-6 Se-2 Corynebacterium minutissimum ATCC 23348 Cm-1Corynebacterium xerosis JCM 1324 Cx-2 Malassezia furfur IFO 0656 Mf-1Staphylococcus aureus IFO 12732 Sa-3 Bacillus subtilis PCI 219 IFO 3134Bs-1 Anaerobic Microorganisms: Propinibadterium Acnes ATCC 12818 Pa-1streptococcus mutans JCM 5157 Su-1

TABLE 10 Results of the anti-bacterial test (MIC: ppm) Test Micro-Sample organ- Acid ism Mix- Compound Compound Compound Compound codeture 1 4 7 10 Se-1 12.5 6.3 12.5 6.3 6.3 Se-2 12.5 6.3 12.5 6.3 6.3 Cm-112.5 6.3 6.3 12.5 6.3 Cx-2 12.5 12.5 12.5 12.5 6.3 Mf-1 6.3 6.3 6.3 6.36.3 Sa-3 12.5 12.5 12.5 12.5 6.3 Bs-1 12.5 6.3 12.5 12.5 6.3 Pa-1 NT12.5 NT NT 12.5 Su-1 NT 6.3 NT NT 6.3 In this table, NT means that thetest was not conducted.

As shown in Table 10, the free acids as the compounds of the inventionexhibited strong activity toward causative bacteria for body odors(Se-1, Se-2), for dandruff (Mf-1), for acne (Pa-1), for caries (Su-1)etc.

Example 5

The melanin production inhibitor of the present invention was used toprepare face lotion, milky lotion, cream, pack, a bath additive andcream foundation respectively.

(1) Face Lotion

TABLE 11 Incorporation amount Ingredients (% by weight) Conc. Glycerin3.0 1,3-Butylene glycol 2.0 Polyoxyethylene sorbitan 1.0 monolaurateEthanol 5.0 Perfume suitable amount Acid mixture (Example 1) 1.0Preservative suitable amount Purified water Adjusted to 100%

(2) Milky Lotion

TABLE 12 Incorporation amount Ingredients (% by weight) Squalane 5.0Vaseline 2.0 Beeswax 0.5 Sorbitan sesquioleate 0.8 Polyoxyethylene oleylether (20 E.O.) 1.2 Compound 1 (Example 2) 0.5 Perfume suitable amountPreservative suitable amount Humectant (propylene glycol) 5.0 Ethanol5.0 Viscous material 20.00 (1.0% aq. Carboxyvinyl polymer) Alkali(potassium hydroxide) 0.1 Purified Water Adjusted to 100%

(3) Cream

TABLE 13 Incorporation amount Ingredients (% by weight) Squalane 5.0Vaseline 2.0 Beeswax 0.5 Sorbitan sesquioleate 0.8 Polyoxyethylene oleylether (20 E.O.) 1.2 Perfume suitable amount Ethyl ester mixture(Example 1) 1.0 Preservative suitable amount Humectant (propyleneglycol) 5.0 Ethanol 5.0 Viscous material 20.0 (1.0% aq. Carboxyvinylpolymer) Alkali (potassium hydroxide) 0.1 Purified Water Adjusted to100%

(4) Pack

TABLE 14 Incorporation amount Ingredients (% by weight) Polyvinylalcohol 15.0 Carboxymethylcellulose sodium 5.0 Propylene glycol 3.0Ethanol 10.0 Perfume Composition suitable amount Compound 10 (Example 3)1.0 Preservative and antioxidant suitable amount Purified water Adjustedto 100%

(5) Bath Additive (Granular Type)

TABLE 15 Incorporation Amount Ingredients (% by weight) Sodium sulfate45.0 Sodium bicarbonate 51.5 Borax 2.0 Carboxymethylcellulose sodium 1.0Pigments suitable amount Perfume suitable amount Compound 4 (Example 2)0.5

(6) Cream Foundation

TABLE 16 Incorporation Amount Ingredients (% by weight) Stearic Acid 5.0Lipophilic glycerin monostearate 2.5 Cetostearyl alcohol 1.0 Propyleneglycol monolaurate 3.0 Fluidic paraffin 7.0 Isopropyl myristate 8.0Butyl paraoxybenzoate suitable amount Triethanolamine 1.2 Sorbit 3.0Methyl p-oxybenzoate suitable amount Titanium oxide 8.0 Kaolin 5.0 Talk2.0 Bentonite 1.0 Coloring pigment suitable amount Acid mixture(Example 1) 1.0 Purified Water Adjusted to 100%

According to the present invention, it was revealed that purifiedproducts of extracts from specific plants have excellent inhibitoryactivity on production of melanin, cell-activating activity, andanti-bacterial activity. The melanin inhibitor consisting of thispurified product is excellent not only as a skin whitening agent forpreventing the formation of stains and freckles, as well as depositionof pigment in the skin after sunburn and for improving appearances, butalso superior in safety and product stability. Further, this purifiedproduct can be used as an agent for external application onto the skin,which is effective for prevention and treatment of body odors, dandruff,wounds, etc. by activating skin cells themselves and activating thefunctions of the skin itself to improve skin conditions. Thesebiologically active substances can be incorporated into base cosmeticssuch as cream, lotion, milky lotion, pack, etc., make-up cosmetics suchas foundation, etc., bath additive, agent for external application ontothe skin, oral cavity composition (e.g., toothpaste, mouthwash), etc.

What is claimed is:
 1. A method of activating cell growth in a humanrequiring such activation comprising applying to the skin of a human acell growth activating amount of a compound represented by formula (1)

where R¹ is —CH₂OH, —COOR⁶ or —COOX, where X is a group capable offorming a salt, R⁶ is hydrogen or C₁ to C₃ lower alkyl and R² to R⁵ eachrepresent hydrogen or methyl; and . . . A . . . is ═C(CH₃)—, —C(CH₃)═,—C(═CH₂)—, —CH(CH₃)— or —C(OH)(CH₃)—.
 2. The method of claim 1, whereinthe compound of formula (1) is selected from the group consisting oflabdanolic acid, labd-7-en-15-oic acid, labd-8(17)-en-15-oic acid,labd-8-en-15-oic acid and salts and methyl and ethyl esters thereof.